中文摘要：

目的分析重组沙门菌表达的结核分枝杆菌（Mycobacterium tuberculosis，Mtb）分泌性蛋白ESAT-6诱导的特异性免疫应答。方法将ESAT-6蛋白编码基因导入原核表达载体pYA3333中，构建重组质粒pYA33-esat。通过电穿孔法转化减毒鼠伤寒沙门菌X4550，获得重组菌X4550（33-esat）。以每只10。CFU剂量的重组菌滴鼻免疫C57BL／6小鼠，间隔18d，在第2次免疫后10d取免疫小鼠脾脏、肺脏、肠系膜淋巴结（mesenteric lymph node，MLN）及派伊尔淋巴集结（Peyer‘s patch，PP）细胞，以ESAT-6多肽作为刺激原，检测特异性的IFN-γ分泌细胞和IL-4分泌细胞。同时，运用CFSE方法检测了体内抗原特异性CTL效应。结果经沙门菌表达并运送的Mtb抗原ESAT-6能诱导特异性的免疫应答。在肺脏及PP细胞中，检测到较高水平的IFN一1和IL_4分泌细胞，免疫应答以Th1型为主。而在脾脏和MLN中，免疫应答呈现Th1／Th2混合应答。此外，体内CTL试验表明，重组菌能够诱导抗原特异的CTL效应，且特异性杀伤率为69．9％。结论以滴鼻方式接种重组沙门菌，不仅能够诱导ESAT-6蛋白特异性的细胞免疫应答，还能激发特异的CTL效应，为结核病的防控提供了新的认识。

英文摘要：

Objective To determine the immune responses induced by recombinant Salmonella typhimurium expressing the secreting antigen ESAT-6 of Mycobacterium tuberculosis. Methods ESAT-6 coding gene was cloned and identified by PCR and sequencing. Prokaryotic expression plasmid pYA33-esat carrying the ESAT-6 coding sequence was constructed firstly and electro-transformed into an attenuated strain X4550 of Salmonella typhimurium, the recombinant bacteria was named as X4550（33-esat）. C57BL/6 mice were immunized intranasally （i. n） with 10s CFU recombinant bacteria at day 0 and 18. Cells from spleen, lung, mesenterie lymph node （MLN） and Peyer＇s patch （PP） were collected from mice after second immunization, and the specific IFN-γ-secreting cells and IL-4-secreting cells were detected by ELISPOT assay using ESAT-6 peptide as stimulus. Furthermore, CTL effects were in vivo evaluated by CFSE assay. Results The results showed that cellular immune responses specific for ESAT-6 could be detected by ELISPOT assay. In lung and PP cells, immune responses against ESAT-6 were biased toward Thl type, the frequency of IFN-γ-secreting cells was much higher than that of IL-4-secreting ceils. In splenocytes and MLN cells, the antigen specific immune responses acted as Thl and Th2 balance, the frequency of IFN-γ-secreting cells was close to that of IL-4-secreting cells. CFSE assay indicated that recombinant bacteria could induce the high level of CTL effects specific for ESAT-6 peptide. Conclusion These results suggested that recombinant Salmonella typhimurium ）（4550 （33-esat） not only can induce cellular immune responses, but also can elicit specific CTL responses after i. n immunization. It also provided the useful information for the control of infectious disease of tuberculosis.